Project Summary/Abstract In order to double all biomass components and support cell division, proliferating cancer cells utilize specialized metabolic programs that affect reliance on particular nutrients. Understanding how the availability of specific nutrients regulates these biosynthetic networks therefore has great potential for developing cancer treatment strategies. In particular, production of nucleotides is critical for proliferating cells, and nucleotide synthesis is the target of many chemotherapies used in the clinic. To gain insight into the metabolic liabilities of nucleotide production, we assessed how nutrient availability affects nucleotide levels in cancer cells. Strikingly, we found that availability of extracellular serine is necessary for the production of nucleotides to support proliferation, even in cancer cells with upregulated intracellular serine synthesis. Serine can provide carbon substrates for nucleotides, but why the source of serine is important for nucleotide production is unclear. In considering this question, we noted that serine synthesis has a high demand for the oxidized cofactor NAD+, whereas direct consumption of serine does not. Indeed, decreasing NAD+ availability prevents serine synthesis and kills cells in the absence of exogenous serine, while enhancing NAD+ regeneration restores nucleotide synthesis and proliferation when extracellular serine is not available. This suggests that cellular redox state can regulate serine synthesis and subsequent use for nucleotide production. Further, dietary interventions to decrease plasma levels of serine inhibit tumor growth in mouse models, suggesting that environmental serine availability may create targetable liabilities in cancer. The overall goal of this project is to determine the role of serine source in regulating nucleotide metabolism to impact cancer cell proliferation. To address this question, I propose to: 1) Define how cellular redox state regulates serine synthesis and nucleotide production in tumors 2) Assess how nucleobase salvage compensates for nucleotide synthesis to support proliferation under serine deprivation 3) Determine how serine availability affects response to chemotherapies that target nucleotide metabolism By addressing these aims, I will gain insight into how environmental nutrient availability regulates nucleotide production in proliferating cells. Importantly, this will inform new strategies to target nucleotide metabolism for cancer therapy. Work on this project will also benefit my training to become a successful independent researcher. During this time, I will continue to develop in my career by attending and presenting my work at seminars and conferences, publishing first author papers, and mentoring younger students. Ultimately, work on this study will train me in addressing the long-term research interests I will pursue in my career: how fundamental biochemical processes affect the biology of cancer.